Human FGF-21 nucleic acids

ABSTRACT

This invention relates to human fibroblast growth factor (hFGF-21), and to variants thereof and to polynucleotides encoding FGF-21. The invention also relates to diagnostic and therapeutic agents related to the polynucleotides and proteins, including probes and antibodies, and to methods of treating liver disease such as cirrhosis and cancer, methods of treating conditions related to thymic function, and methods of treating conditions of the testis. The invention also relates to mouse fibroblast growth factor (mFGF-21), and to variants thereof and polynucleotides encoding mFGF-21.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional PatentApplication No. 60/166,540 filed Nov. 18, 1999 and U.S. ProvisionalPatent Application No. 60/203,633 filed May 11, 2000, which areincorporated by reference herein in their entirety.

TECHNICAL FIELD

The present invention relates to nucleic acid sequences encoding amember of the fibroblast growth factor (FGF) family, and to polypeptidesencoded by the nucleic acid sequence.

BACKGROUND OF THE INVENTION

The prototypic fibroblast growth factors (FGFs), FGF-1 and FGF-2, wereoriginally isolated from brain and pituitary as mitogens forfibroblasts. However, FGF-1 and FGF-2 are widely expressed in developingand adult tissues, and are polypeptides with multiple biologicalactivities including angiogenesis, mitogenesis, cellular differentiationand repair of tissue injury (Baird, A. et al., Cancer Cells 3:239-243(1991); Burgess, W. H. et al., Annu. Rev. Biochem. 58:575-606 (1989).According to the published literature, the FGF family now consists of atleast nineteen members, FGF-1 to FGF-19. FGF-3 was identified to be acommon target for activation by the mouse mammary tumor virus (Dicksonet al., Ann. N.Y. Acad. Sci. 638:18-26 (1991); FGF-4 to FGF-6 wereidentified as oncogene products (Yoshida et al., Ann. NY Acad. Sci.638:27-37 (1991); Goldfarb et al., Ann. NY Acad. Sci 638:38-52 (1991);Coulier et al., Ann. NY Acad. Sci. 638:53-61 (1991)). FGF-10 wasidentified from rat lung by homology-based polymerase chain reaction(PCR) (Yamasaki et al., J. Biol. Chem. 271:15918-15921 (1996)). FGF-11to FGF-14 (FGF homologous factors (FHFs) 1 to 4) were identified fromhuman retina by a combination of random cDNA sequencing, data basesearches and homology-based PCR (Smallwood et al., Proc. Natl. Acad.Sci. USA 93:9850-9857 (1996)). FGF-15 was identified as a downstreamtarget of a chimeric homeodomain oncoprotein (McWhirter et al.,Development 124:3221-3232 (1997)). FGF-16, FGF-17, and FGF-18 wereidentified from rat heart and embryos by homology-based PCR,respectively (Miyake et al., Biochem. Biophys. Res. Commun. 243:148-152(1998); Hoshikawa et al., Biochem. Biophys. Res. Commun. 244:187-191(1998); Ohbayashi et al., J. Biol. Chem. 273:18161-18164 (1998)).Recently, FGF-19 was identified from human fetal brain by data basesearch (Nishimura et al., Biochim. Biophys. Acta 1444:148-151 (1999)).They have a conserved ˜120-amino acid residue core with ˜30 to 60% aminoacid identity. These FGFs also appear to play important roles in bothdeveloping and adult tissues. Thus, there is a need in the art foradditional FGF molecules having functions and activities that differfrom the known FGFs and for FGF molecules specifically expressed intissues implicated in human disease.

SUMMARY OF THE INVENTION

The present invention provides a composition comprising an isolatedpolynucleotide selected from the group consisting of:

(a) a polynucleotide comprising at least eight contiguous nucleotides ofSEQ ID NO:1 or 3;

(b) a polynucleotide having at least 80% homology to the polynucleotideof (a); and

(c) a polynucleotide encoding a protein expressed by a polynucleotidehaving the sequence of SEQ ID NO:1 or 3.

The invention further provides for the use of the isolatedpolynucleotides or fragments thereof as diagnostic probes or as primers.

The present invention also provides a composition comprising apolypeptide, wherein said polypeptide is selected from the groupconsisting of:

(a) a polypeptide comprising at least 6 contiguous amino acids encodedby SEQ ID NO:1 or 3;

(b) a polypeptide encoded by a polynucleotide comprising SEQ ID NO:1 or3; and

(c) a variant of the polypeptide of SEQ ID NO:2 or 4.

In certain preferred embodiments of the invention, the polynucleotide isoperably linked to an expression control sequence. The invention furtherprovides a host cell, including bacterial, yeast, insect and mammaliancells, transformed with the polynucleotide sequence. The invention alsoprovides full-length cDNA and full-length polynucleotides correspondingto SEQ ID NO:1 or 3.

Protein and polypeptide compositions of the invention may furthercomprise a pharmaceutically acceptable carrier. Compositions comprisingan antibody that specifically reacts with such protein or polypeptideare also provided by the present invention.

The invention also provides for the production of large amounts ofotherwise minor cell populations of cells to be used for generation ofcDNA libraries for the isolation of rare molecules expressed in theprecursors cells or progeny; cells produced by treatment may directlyexpress growth factors or other molecules, and conditioned media isscreened in assays for novel activities.

The invention further provides for the isolation, self-renewal andsurvival of mammalian stem cells and the differentiation of theirprogeny.

The invention also provides for compositions and methods of preventingor slowing the degeneration of or increasing the numbers of hepaticcells, in disease states including but not limited to, cirrhosis of theliver, hepatitis, and post-surgical and post-injury tissue regeneration;of preventing or slowing degeneration of or increasing the numbers ofcells in the testes in disease states such as infertility and impotence,and of preventing or slowing degeneration of or increasing the numbersof cells of the thymus in disorders of the thymus and immune system.

The invention also provides for compositions and methods for identifyinginhibitors of FGF-21 function, useful in disease states such as liverand testicular cancers, or leukemias, lymphomas or other cancers, andproliferative or differentiation disorders of cells derived from thethymus.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Amino acid sequence comparison of human FGF-21 (SEQ ID NO: 4)with mouse FGF-15 (SEQ ID NO: 9). Asterisks indicate identical aminoacid residues of the sequences.

FIG. 2. Amino acid sequence comparison of human FGF-21 (SEQ ID NO: 4)and human FGF-19 (SEQ ID NO: 10). Asterisks indicate identical aminoacid residues of the sequence.

FIG. 3. Expression of FGF-21 in mouse tissues.

FIGS. 4A and 4B. DNA sequence (SEQ ID NO:1) and amino acid sequence (SEQID NO:2) of mouse FGF-21.

FIGS. 5A and 5B. DNA sequence (SEQ ID NO:3) and amino acid sequence (SEQID NO:4) of human FGF-21.

FIG. 6. Alignment of the amino acid sequences of human (SEQ ID NO:4) andmouse (SEQ ID NO:2) FGF-21.

FIGS. 7A and 7B. FIGS. 7A and 7B provides codon usage for yeast. Thefirst field of information on each line of the table contains athree-letter code for an amino acid. The second field contains anunambiguous codon for that amino acid. The third field lists the numberof occurrences of that codon in the genes from which the table iscompiled. The fourth field lists the expected number of occurrences ofthat codon per 1,000 codons in genes whose codon usage is identical tothat compiled in the codon frequency table. The last field contains thefraction of occurrences of the codon in its synonymous codon family.

FIGS. 8A and 8B. FIGS. 8A and 8B provides codon usage for Drosophila.

FIGS. 9A and 9B. FIGS. 9A and 9B provides codon usage for E. coli.

DETAILED DESCRIPTION OF THE INVENTION

Because of their potent activities for promoting growth, proliferation,survival and differentiation of a wide variety of cells and tissuetypes, FGFs continue to be pursued as therapeutic agents for a number ofdifferent indications, including wound healing, such as musculo-skeletalconditions, for example, bone fractures, ligament and tissue repair,tendonitis, bursitis, etc.; skin conditions, for example, burns, cuts,lacerations, bed sores, slow healing ulcers, etc.; tissue protection,repair, and the induction of angiogenesis during myocardial infarctionand ischemia, in the treatment of neurological conditions, for example,neuro-degenerative disease and stroke, in the treatment of eye disease,including macular degeneration, and the like.

The fibroblast growth factor (FGF) proteins identified to date belong toa family of signaling molecules that regulate growth and differentiationof a variety of cell types. The significance of FGF proteins to humanphysiology and pathology relates in part to their key roles inembryogenesis, in blood vessel development and growth, and in bonegrowth. In vitro experiments have demonstrated a role for FGF inregulating cell growth and division of endothelial cells, vascularsmooth muscle cells, fibroblasts, and cardiac and skeletal myocytes.Other members of the FGF family and their biological roles are describedin Crossley et al., Development 121:439-451 (1995); Ohuchi et al.,Development 124:2235-2244 (1997); Gemel et al., Genomics 35:253-257(1996); and Ghosh et al., Cell Growth and Differentiation 7:1425-1434(1996).

FGF proteins are also significant to human health and disease because ofa role in cancer cell growth. For example, FGF-8 was identified as anandrogen-induced growth factor in breast and prostate cancer cells.(Tanaka et al., FEBS Lett. 363:226-230 (1995) and P.N.A.S. 89:8928-8932(1992)).

The role of FGF in normal development is being elucidated in partthrough studies of FGF receptors. Wilke, T. et al., Dev. Dynam.210:41-52 (1997) found that FGFR1, FGFR2, and FGFR3 transcripts werelocalized to specific regions of the head during embryonic developmentin chickens. The expression pattern correlated with areas affected byhuman FGFR mutations in Crouzon syndrome, a condition of abnormalintramembranous bone formation. Belluardo, N. et al., Jour. Comp. Neur.379:226-246 (1997) studied localization of FGFR 1, 2, and 3 mRNAs in ratbrain, and found cellular specificity in several brain regions.Furthermore, FGFR1 and FGFR2 mRNAs were expressed in astroglial reactivecells after brain lesion, supporting a role of certain FGF's in braindisease and injury. Ozawa, K. et al., Mol. Brain Res. 41:279-288 (1996)reported that FGF1 and FGF-5 expression increased after birth, whereasFGF3, FGF-6, FGF-7, and FGF-8 genes showed higher expression in lateembryonic stages than in postnatal stages.

New members of the FGF family are described here, wherein the FGFprotein is expressed in a variety of tissues but most abundantly in theliver. A polynucleotide encoding the mouse FGF of the invention has thesequence as shown in SEQ ID NO:1. A polynucleotide encoding the humanFGF of the invention has the sequence as shown in SEQ ID NO:3. The mousepolynucleotide was identified as encoding a member of the FGF family bythe conserved regions throughout the amino acid sequence and by theregions of homology shared by the polynucleotide and genes encodingknown FGF proteins.

The inventors believe that FGF-21 is a previously unidentified member ofthe FGF family. To date, over 19 human FGF proteins have beenidentified. In most cases, homologous proteins in other mammals,particularly mice and rats, have also been identified. The humanproteins vary to different degrees in terms of amino acid sequence,receptor specificity, tissue expression patterns, and biologicalactivity.

The present FGF-21 differs in sequence from all the FGF proteinsdescribed to date in publications. As discussed herein, the knowledgeabout the roles played by various FGF proteins continues to grow, but isby far incomplete.

The present invention adds to this knowledge by disclosing that the FGFof SEQ ID NO:1 is highly expressed in liver, and human FGF-21 may play arole in development of and recovery from liver disease. Further, FGF-21is also expressed in testis and thymus, and therefore may play a role inthe development or recovery from disorders of testicular function orfunction of cells derived from the thymus. The invention therefore isbased upon the identification, isolation and sequencing of a newfibroblast growth factor (FGF-21).

Isolation and Analysis of Mouse cDNA Encoding FGF-21

According to the invention, DNA encoding a novel mouse FGF has beenidentified. The nucleotide sequence of the entire coding region wasdetermined by adaptor-ligation mediated polymerase chain reaction usingmouse embryo cDNA as a template. The nucleotide sequence of the codingregion allowed for the elucidation of the complete amino acid sequenceof the mouse FGF (210 amino acids) (FIG. 4). This protein is tentativelynamed FGF-21.

Isolation and Analysis of Human cDNA Encoding FGF-21

A human gene encoding FGF-21 was located in the 5′ flanking region of aputative human α-fucosyltransferase gene. The cDNA encoding the entirecoding region of human FGF-21 was amplified from fetal brain cDNA by PCRusing FGF-specific primers as follows: sense primer: 5′agccattgatggactcggac 3′ (SEQ ID NO:5); antisense primer: 5′tggcttcaggaagcgtagct 3′ (SEQ ID NO:6).

Expression of FGF-21 mRNA in Adult Mouse Tissues

The expression of FGF-21 mRNA was examined in adult mouse major tissuesincluding brain, heart, lung, liver, kidney, spleen, lung, thymus,testis, muscle, skin, and small intestine by polymerase chain reaction.FGF-21 mRNA expression was detected at high levels in the liver (FIG.3). Expression was also seen in testis and thymus. To confirm theexpression of FGF-21 mRNA in mouse tissues, mouse tissue (A)⁺RNA wasexamined by Northern blotting analysis using a ³²P-labeled rat FGF-21cDNA probe. The results confirmed a high level of expression in mouseliver. Expression was also seen in thymus; larger transcripts were seenin testis tissue.

Reference to FGF-21 herein is intended to be construed to include growthfactors of any origin which are substantially homologous to and whichare biologically equivalent to the FGF-21 characterized and describedherein. Such substantially homologous growth factors may be native toany tissue or species and, similarly, biological activity can becharacterized in any of a number of biological assay systems.

The term “biologically equivalent” is intended to mean that thecompositions of the present invention are capable of demonstrating someor all of the same growth properties in a similar fashion, notnecessarily to the same degree as the FGF-21 isolated as describedherein or recombinantly produced human FGF-21 of the invention.

By “substantially homologous” it is meant that the degree of homology ofhuman FGF-21 to FGF-21 from any species is greater than that betweenFGF-21 and any previously reported member of the FGF family.

Sequence identity or percent identity is intended to mean the percentageof same residues between two sequences, referenced to human FGF whendetermining percent identity with non-human FGF-21, referenced to FGF-21when determining percent identity with non-FGF-21 growth factors, whenthe two sequences are aligned using the Clustal method (Higgins et al,Cabios 8:189-191, 1992) of multiple sequence alignment in the Lasergenebiocomputing software (DNASTAR, INC, Madison, Wis.). In this method,multiple alignments are carried out in a progressive manner, in whichlarger and larger alignment groups are assembled using similarity scorescalculated from a series of pairwise alignments. Optimal sequencealignments are obtained by finding the maximum alignment score, which isthe average of all scores between the separate residues in thealignment, determined from a residue weight table representing theprobability of a given amino acid change occurring in two relatedproteins over a given evolutionary interval. Penalties for opening andlengthening gaps in the alignment contribute to the score. The defaultparameters used with this program are as follows: gap penalty formultiple alignment=10; gap length penalty for multiple alignment=10;k-tuple value in pairwise alignment=1; gap penalty in pairwisealignment=3; window value in pairwise alignment=5; diagonals saved inpairwise alignment=5. The residue weight table used for the alignmentprogram is PAM250 (Dayhoff et al., in Atlas of Protein Sequence andStructure, Dayhoff, Ed., NDRF, Washington, Vol. 5, suppl. 3, p. 345,1978).

Percent conservation is calculated from the above alignment by addingthe percentage of identical residues to the percentage of positions atwhich the two residues represent a conservative substitution (defined ashaving a log odds value of greater than or equal to 0.3 in the PAM250residue weight table). Conservation is referenced to human FGF-21 whendetermining percent conservation with non-human FGF-21, and referencedto FGF-21 when determining percent conservation with non-FGF-21 growthfactors. Conservative amino acid changes satisfying this requirementare: R-K; E-D, Y-F, L-M; V-I, Q-H.

The invention provides FGF-21 proteins or variants thereof having one ormore polymers covalently attached to one or more reactive amino acidside chains. By way of example, not limitation, such polymers includepolyethylene glycol (PEG), which can be attached to one or more freecysteine sulfhydryl residues, thereby blocking the formation ofdisulfide bonds and aggregation when the protein is exposed to oxidizingconditions. In addition, pegylation of FGF-21 proteins and/or muteins isexpected to provide such improved properties as increased half-life,solubility, and protease resistance. FGF-21 proteins and/or muteins mayalternatively be modified by the covalent addition of polymers to freeamino groups such as the lysine epsilon or the N-terminal amino group.Preferred cysteines and lysines for covalent modification will be thosenot involved in receptor or heparin binding or in proper proteinfolding. For example, cys 27 and cys 104 may be modified. It will beapparent to one skilled in the art that the methods for assaying FGF-21biochemical and/or biological activity may be employed in order todetermine if modification of a particular amino acid residue affects theactivity of the protein as desired.

It may be advantageous to improve the stability of FGF-21 by modifyingone or more protease cleavage sites. Thus, the present inventionprovides FGF-21 variants in which one or more protease cleavage site hasbeen altered by, for example, substitution of one or more amino acids atthe cleavage site in order to create an FGF-21 variant with improvedstability. Such improved protein stability may be beneficial duringprotein production and/or therapeutic use. A preferred site is amonobasic site within two residues of a proline, such as near residue160 of SEQ ID NO:4.

Suitable protease cleavage sites for modification are well known in theart and likely will vary depending on the particular applicationcontemplated. For example, typical substitutions would includereplacement of lysines or arginines with other amino acids such asalanine. The loss of activity, such as receptor binding or heparinbinding, can be tested for as described herein.

FGF-21 can also include hybrid and modified forms of FGF-21 includingfusion proteins and FGF-21 fragments and hybrid and modified forms inwhich certain amino acids have been deleted or replaced andmodifications such as where one or more amino acids have been changed toa modified amino acid or unusual amino acid and modifications such asglycosylations so long as the hybrid or modified form retains thebiological activity of FGF-21. Fusion proteins can consist of the FGF-21of the invention or fragment thereof and a signal sequence of aheterologous protein to promote secretion of the protein product.

Fusion proteins comprising FGF-21 or a biologically active or antigenicfragment thereof can be produced using methods known in the art. Suchfusion proteins can be used therapeutically or can be produced in orderto simplify the isolation and purification procedures. Histidineresidues can be incorporated to allow immobilized metal affinitychromatography purification. Residues EQKLISEEDL (SEQ ID NO: 11) containthe antigenic determinant recognized by the myc monoclonal antibody andcan be incorporated to allow myc monoclonal antibody-based affinitypurification. A thrombin cleavage site can be incorporated to allowcleavage of the molecule at a chosen site; a preferred thrombin cleavagesite consists of residues LVPRG (SEQ ID NO: 12). Purification of themolecule can be facilitated by incorporating a sequence, such asresidues SAWRHPQFGG (SEQ ID NO: 13), which binds to paramagneticstreptavidin beads. Such embodiments are described in WO 97/25345, whichis incorporated by reference.

The invention further includes chimeric molecules between FGF-21 andkeratinocyte growth factor (KGF) (Reich-Slotky, R. et al., J. Biol.Chem. 270:29813-29818 (1995)). The chimeric molecule can containspecific regions or fragments of one or both of the FGF-21 and KGFmolecules, such as the FGF-21 fragments described below.

The invention also includes fragments of FGF-21. Preferred fragments ofSEQ ID NO:4 and 2, respectively, include: amino acids from about 1 toabout 209 (210 for SEQ ID NO:2); amino acids from about 2 to about 209(210 for SEQ ID NO:2); amino acids from about 1 to about 177; aminoacids from about 40 to about 209 for SEQ ID NO:2 and amino acids fromabout 40 to about 177. Such fragments can be prepared from the proteinsby standard biochemical methods, or by expressing a polynucleotideencoding the fragment.

FGF-21, or a fragment thereof, can be produced as a fusion proteincomprising human serum albumin (HSA) or a portion thereof. Such fusionconstructs are suitable for enhancing expression of the FGF-21, orfragment thereof, in an eukaryotic host cell. Exemplary HSA portionsinclude the N-terminal polypeptide (amino acids 1-369, 1-419, andintermediate lengths starting with amino acid 1), as disclosed in U.S.Pat. No. 5,766,883, and publication WO 97/24445, incorporated byreference herein. Other chimeric polypeptides can include a HSA proteinwith FGF-21, or fragments thereof, attached to each of the C-terminaland N-terminal ends of the HSA. Such HSA constructs are disclosed inU.S. Pat. No. 5,876,969, incorporated by reference herein.

Also included with the scope of the invention are FGF-21 molecules thatdiffer from native FGF-21 by virtue of changes in biologically activesites.

Growth factors are thought to act at specific receptors. According tothe invention, FGF-21 and as yet unknown members of this family ofgrowth factors act through specific receptors having distinctdistributions as has been shown for other growth factor families.

A preferred hFGF-21 of the present invention has been identified. Alsopreferred is hFGF-21 prepared by recombinant DNA technology. Includedwithin the scope of the invention are polynucleotides, including DNA andRNA, with 80% homology to SEQ ID NO:1 or SEQ ID NO:3; preferably atleast 85% homology, more preferably at least 90% homology, mostpreferably 95% homology. Polynucleotides with 96%, 97%, 98%, and 99%homology to SEQ ID NO:1 or 3 are also included. Percent homology iscalculated using methods known in the art. A non-limiting example ofsuch a method is the Smith-Waterman homology search algorithm asimplemented in MPSRCH program (Oxford Molecular), using an affine gapsearch with a gap open penalty of 12 and a gap extension penalty of 1.

FGF-21 can also include hybrid and modified forms of FGF-21 includingfusion proteins and FGF-21 fragments and hybrid and modified forms inwhich certain amino acids have been deleted or replaced andmodifications such as where one or more amino acids have been changed toa modified amino acid or unusual amino acid and modifications such asglycosylations so long as the hybrid or modified form retains thebiological activity of FGF-21. By retaining the biological activity, itis meant that the ability of FGF-21 to promote the growth, survival ordifferentiation of responsive cells is preserved, although notnecessarily at the same level of potency as that of the FGF-21 isolatedas described herein or that of the recombinantly produced FGF-21.

Also included within the meaning of substantially homologous is anyFGF-21 which may be isolated by virtue of cross-reactivity withantibodies to the FGF-21 described herein or whose encoding nucleotidesequences including genomic DNA, mRNA or cDNA may be isolated throughhybridization with the complementary sequence of genomic or subgenomicnucleotide sequences or cDNA of the FGF-21 herein or fragments thereof.It will also be appreciated by one skilled in the art that degenerateDNA sequences can encode human FGF-21 and these are also intended to beincluded within the present invention as are allelic variants of FGF-21.

Recombinant human FGF-21 may be made by expressing the DNA sequencesencoding FGF-21 in a suitable transformed host cell. Using methods wellknown in the art, the DNA encoding FGF-21 may be linked to an expressionvector, transformed into a host cell and conditions established that aresuitable for expression of FGF-21 by the transformed cell.

The DNA encoding FGF-21 can be engineered to take advantage of preferredcodon usage of host cells. Codon usage in Pseudomonas aeruginosa isdescribed in, for example, West et al., Nucleic Acids Res. 11:9323-9335(1988). Codon usage in Saccharomyces cerevisiae is described in, forexample, Lloyd et al., Nucleic Acids Res. 20:5289-5295 (1992). Codonpreference in Corynebacteria and a comparison with E. coli preference isprovided in Malubres et al., Gene 134:15-24 (1993). Codon usage inDrosophila melanogaster is described in, for example, Akashi, Genetics136:927-935 (1994). Codon usage in yeast is also shown in FIG. 7, codonusage in Drosophila is shown in FIG. 8, and codon usage for E. coli isshown in FIG. 9.

Any suitable expression vector may be employed to produce recombinanthuman FGF-21 such as expression vectors for use in insect cells.Baculovirus expression systems can also be employed. A preferable methodis expression in insect cells, such as Tr5 or Sf9 cells, usingbaculovirus vector.

The present invention includes nucleic acid sequences includingsequences that encode human FGF-21. Also included within the scope ofthis invention are sequences that are substantially the same as thenucleic acid sequences encoding FGF-21. Such substantially the samesequences may, for example, be substituted with codons more readilyexpressed in a given host cell such as E. coli according to well knownand standard procedures. Such modified nucleic acid sequences areincluded within the scope of this invention.

Specific nucleic acid sequences can be modified by those skilled in theart and, thus, all nucleic acid sequences that code for the amino acidsequences of FGF-21 can likewise be so modified. The present inventionthus also includes nucleic acid sequence which will hybridize with allsuch nucleic acid sequences, or complements of the nucleic acidsequences where appropriate, and encode a polypeptide having the cellsurvival, growth or differentiation activity of FGF-21. The presentinvention also includes nucleic acid sequences that encode polypeptidesthat have cell survival promoting activity and that are recognized byantibodies that bind to FGF-21. Preferred methods and epitopes forraising antibodies are described in Example 4.

The present invention also encompasses vectors comprising expressionregulatory elements operably linked to any of the nucleic acid sequencesincluded within the scope of the invention. This invention also includeshost cells of any variety that have been transformed with vectorscomprising expression regulatory elements operably linked to any of thenucleic acid sequences included within the scope of the presentinvention.

Methods are also provided herein for producing FGF-21. Preparation canbe by isolation from conditioned medium from a variety of cell types solong as the cell type produces FGF-21. A second and preferred methodinvolves utilization of recombinant methods by isolating or obtaining anucleic acid sequence encoding FGF-21, cloning the sequence along withappropriate regulatory sequences into suitable vectors and cell types,and expressing the sequence to produce FGF-21.

Although FGF-21 has been described on the basis of its high expressionlevel in liver, this factor may act on other cell types as well. It islikely that FGF-21 will act on non-liver cells to promote theirsurvival, growth, differentiation state or function. This expectation isbased upon the activity of known growth factors. Members of the FGFfamily act on many cell types of different function and embryologicorigin, even when their expression is limited to one or a few tissues.

The inventors herein have identified that FGF-21 is expressed at ahigher level in liver. This suggests a role for FGF-21 in, for example,precancerous lesions, hepatoma, cirrhosis, repair, from inflammatorydiseases, trauma or other types of injury, and other diseases of theliver. Further, FGF-21 is also expressed in thymus and testis. Thissuggests a role for FGF-21 in, for example, infertility, control oftestosterone production, cancer of the testis or associated cells, andother disorders of the testis, and in disorders of cells such as immunecells derived from the thymus, for example, autoimmune disorders,leukemias and lymphomas, immune deficiency states, and the like.

The present invention also includes therapeutic or pharmaceuticalcompositions comprising FGF-21 in an effective amount for treatingpatients with liver, testis or thymic disease, and a method comprisingadministering a therapeutically effective amount of FGF-21. Thesecompositions and methods are useful for treating a number of diseases.The compositions and methods herein can also be useful to preventdegeneration and/or promote survival in other non-liver tissues as well,such as promoting angiogenesis, neuronal survival, wound healing, andthe like. One skilled in the art can readily use a variety of assaysknown in the art to determine whether FGF-21 would be useful inpromoting survival or functioning in a particular cell type. Promotionof neuronal survival is useful in the treatment of nervous systemdiseases and conditions, including Parkinson's disease, Alzheimersdisease, traumatic injury to nerves, and degenerative disease of thenervous system.

In certain circumstances, it may be desirable to modulate or decreasethe amount of FGF-21 expressed. Thus, in another aspect of the presentinvention, FGF-21 anti-sense oligonucleotides can be made and a methodutilized for diminishing the level of expression of FGF-21 by a cellcomprising administering one or more FGF-21 anti-sense oligonucleotides.By FGF-21 anti-sense oligonucleotides reference is made tooligonucleotides that have a nucleotide sequence that interacts throughbase pairing with a specific complementary nucleic acid sequenceinvolved in the expression of FGF-21 such that the expression of FGF-21is reduced. Preferably, the specific nucleic acid sequence involved inthe expression of FGF-21 is a genomic DNA molecule or mRNA molecule thatencodes FGF-21. This genomic DNA molecule can comprise regulatoryregions of the FGF-21 gene, or the coding sequence for mature FGF-21protein. The term complementary to a nucleotide sequence in the contextof FGF-21 antisense oligonucleotides and methods therefor meanssufficiently complementary to such a sequence as to allow hybridizationto that sequence in a cell, i.e., under physiological conditions. TheFGF-21 antisense oligonucleotides preferably comprise a sequencecontaining from about 8 to about 100 nucleotides and more preferably theFGF-21 antisense oligonucleotides comprise from about 15 to about 30nucleotides. The FGF-21 antisense oligonucleotides can also contain avariety of modifications that confer resistance to nucleolyticdegradation such as, for example, modified internucleoside linages(Uhlmann and Peyman, Chemical Reviews 90:543-548 1990; Schneider andBanner, Tetrahedron Left. 31:335, 1990 which are incorporated byreference), modified nucleic acid bases and/or sugars and the like.

The therapeutic or pharmaceutical compositions of the present inventioncan be administered by any suitable route known in the art including forexample intravenous, subcutaneous, intramuscular, transdermal,intrathecal or intracerebral. Administration can be either rapid as byinjection or over a period of time as by slow infusion or administrationof slow release formulation.

FGF-21 can also be linked or conjugated with agents that providedesirable pharmaceutical or pharmacodynamic properties. For example,FGF-21 can be coupled to any substance known in the art to promotepenetration or transport across the blood-brain barrier such as anantibody to the transferring receptor, and administered by intravenousinjection (see, for example, Friden et al., Science 259:373-377, 1993which is incorporated by reference). Furthermore, FGF-21 can be stablylinked to a polymer such as polyethylene glycol to obtain desirableproperties of solubility, stability, half-life and otherpharmaceutically advantageous properties. (See, for example, Davis etal., Enzyme Eng. 4:169-73, 1978; Burnham, Am. J. Hosp. Pharm.51:210-218, 1994 which are incorporated by reference.)

The compositions are usually employed in the form of pharmaceuticalpreparations. Such preparations are made in a manner well known in thepharmaceutical art. One preferred preparation utilizes a vehicle ofphysiological saline solution, but it is contemplated that otherpharmaceutically acceptable carriers such as physiologicalconcentrations of other non-toxic salts, five percent aqueous glucosesolution, sterile water or the like may also be used. It may also bedesirable that a suitable buffer be present in the composition. Suchsolutions can, if desired, be lyophilized and stored in a sterileampoule ready for reconstitution by the addition of sterile water forready injection. The primary solvent can be aqueous or alternativelynon-aqueous. FGF-21 can also be incorporated into a solid or semi-solidbiologically compatible matrix which can be implanted into tissuesrequiring treatment.

The carrier can also contain other pharmaceutically-acceptableexcipients for modifying or maintaining the pH, osmolarity, viscosity,clarity, color, sterility, stability, rate of dissolution, or odor ofthe formulation. Similarly, the carrier may contain still otherpharmaceutically-acceptable excipients for modifying or maintainingrelease or absorption or penetration across the blood-brain barrier.Such excipients are those substances usually and customarily employed toformulate dosages for parenteral administration in either unit dosage ormulti-dose form or for direct infusion into the cerebrospinal fluid bycontinuous or periodic infusion.

Dose administration can be repeated depending upon the pharmacokineticparameters of the dosage formulation and the route of administrationused.

It is also contemplated that certain formulations containing FGF-21 areto be administered orally. Such formulations are preferably encapsulatedand formulated with suitable carriers in solid dosage forms. Someexamples of suitable carriers, excipients, and diluents include lactose,dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calciumphosphate, alginates, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidone, cellulose, gelatin, syrup, methyl cellulose,methyl- and propylhydroxybenzoates, talc, magnesium, stearate, water,mineral oil, and the like. The formulations can additionally includelubricating agents, wetting agents, emulsifying and suspending agents,preserving agents, sweetening agents or flavoring agents. Thecompositions may be formulated so as to provide rapid, sustained, ordelayed release of the active ingredients after administration to thepatient by employing procedures well known in the art. The formulationscan also contain substances that diminish proteolytic degradation andpromote absorption such as, for example, surface active agents.

Depending on the treatment regimen contemplated, it may be desired tocontrol the rate of release of FGF-21 protein or variant thereof toprovide long-term treatment while minimizing the frequency ofadministration. Such treatment regimens may be desired, for example,where the FGF-21 protein is found to be relatively unstable such thatthe localized concentration of active protein is at an efficacious levelfor an insufficient period of time. Thus, for example, for certaindiseases, it may not be desired or practical to perform repeated andfrequent injections. The major advantages of such sustained releasesystems include targeted local delivery of drugs at a constant rate,less drug required to treat the disease state, minimization of possibleside effects, and enhanced efficacy of treatment. Also, these forms ofdelivery systems are capable of protecting drugs that are unstable invivo and that would normally require a frequent dosing interval. Undersuch circumstances, sustained release may be achieved by one of themethods readily available in the art such as the encapsulation of FGF-21conjugated heparin-Sepharose beads to form heparin-alginate microspheresor the preparation of FGF-21 PLG microspheres.

Heparin-alginate microspheres have been successfully employed for thedelivery of Basic Fibroblast Growth Factor to tissue (Lopez et al.,Journal of Pharmacology and Experimental Therapeutics 282(1):385-390(1997)). Similarly, Alginate/heparin-Sepharose microspheres and filmshave been used as drug carriers to control the release of a basicFGF-saponin conjugate in order to control its release in small doses.Addition of heparin to solutions of bFGF prevents losses in activitythat accompany changes in pH or elevation in temperature. See, forexample, Gospodarowicz et al., J. Cell. Physiol. 128:475-484 (1986).

Binding of FGF-21 to heparin may be employed in order to enhance itsstability either during in vivo expression or administration or in vitroduring various stages of protein purification. Thus, by the presentinvention, heparin may be added to a solution of FGF-21 and the activityassayed by the methods disclosed herein.

FGF-21 bound heparin-Sepharose beads may be encapsulated into calciumalginate microspheres to permit the controlled release of theheparin-stabilized FGF-21 protein. For example, microspheres may beconstructed by dropping a mixed solution of sodium alginate with FGF-21bound heparin-Sepharose beads into a hardening solution of calciumchloride. Spheres are formed instantaneously as the mixture enters thehardening solution. The size of the microsphere may be adjusted bypassing the FGF-21 bound heparin-Sepharose beads through a cylinder ofreduced cross-sectional area such as through a hypodermic needle.

Encapsulation efficiency may be determined by comparing the amount ofencapsulated growth factor with that initially present in solution. Forexample, the FGF-21 may be stripped from the heparin-Sepharose beadswith a solution of 3 M NaCl and functional activity assays may beperformed.

The specific dose is calculated according to the approximate body weightor body surface area of the patient or the volume of body space to beoccupied. The dose will also be calculated dependent upon the particularroute of administration selected. Further refinement of the calculationsnecessary to determine the appropriate dosage for treatment is routinelymade by those of ordinary skill in the art. Such calculations can bemade without undue experimentation by one skilled in the art in light ofthe activity disclosed herein in assay preparations of target cells.Exact dosages are determined in conjunction with standard dose-responsestudies. It will be understood that the amount of the compositionactually administered will be determined by a practitioner, in the lightof the relevant circumstances including the condition or conditions tobe treated, the choice of composition to be administered, the age,weight, and response of the individual patient, the severity of thepatient's symptoms, and the chosen route of administration.

In one embodiment of this invention, FGF-21 may be therapeuticallyadministered by implanting into patients vectors or cells capable ofproducing a biologically-active form of FGF-21 or a precursor of FGF-21,i.e., a molecule that can be readily converted to a biological-activeform of FGF-21 by the body. In one approach cells that secrete FGF-21may be encapsulated into semipermeable membranes for implantation into apatient. The cells can be cells that normally express FGF-21 or aprecursor thereof or the cells can be transformed to express FGF-21 or aprecursor thereof. It is preferred that the cell be of human origin andthat the FGF-21 be human FGF-21 when the patient is human. However, theformulations and methods herein can be used for veterinary as well ashuman applications and the term “patient” as used herein is intended toinclude human and veterinary patients.

Cells can be grown ex vivo for use in transplantation or engraftmentinto patients (Muench et al., Leuk. & Lymph. 16:1-11, 1994 which isincorporated by reference). In another embodiment of the presentinvention, FGF-21 is used to promote the ex vivo expansion of a cellsfor transplantation or engraftment. Current methods have used bioreactorculture systems containing factors such as erythropoietin, colonystimulating factors, stem cell factor, and interleukins to expandhematopoietic progenitor cells for erythrocytes, monocytes, neutrophils,and lymphocytes (Verfaillie, Stem Cells 12:466-476, 1994 which isincorporated by reference). These stem cells can be isolated from themarrow of human donors, from human peripheral blood, or from umbilicalcord blood cells. The expanded blood cells are used to treat patientswho lack these cells as a result of specific disease conditions or as aresult of high dose chemotherapy for treatment of malignancy (George,Stem Cells 12(Suppl 1):249-255, 1994 which is incorporated byreference). In the case of cell transplant after chemotherapy,autologous transplants can be performed by removing bone marrow cellsbefore chemotherapy, expanding the cells ex vivo using methods that alsofunction to purge malignant cells, and transplanting the expanded cellsback into the patient following chemotherapy (for review, see Rummel andVan Zant, J. Hematotherapy 3:213-218, 1994 which is incorporated byreference). Since FGF-21 is expressed in liver cells, it is believedthat FGF-21 can function to prevent or slow the progression of cirrhosischanges in liver cells, and to promote hepatic cell regeneration afterinjury or after surgical removal of part of the liver due to disease.

In a number of circumstances it would be desirable to determine thelevels of FGF-21 in a patient. The identification of FGF-21 along withthe present report showing expression of FGF-21 provides the basis forthe conclusion that the presence of FGF-21 serves a normal physiologicalfunction related to cell growth and survival. Endogenously producedFGF-21 may also play a role in certain disease conditions.

Given that FGF-21 is expressed in liver, thymic and testicular tissue,it is likely that the level of FGF-21 may be altered in a variety ofconditions and that quantification of FGF-21 levels would provideclinically useful information. Furthermore, in the treatment ofdegenerative conditions, altered physiological function or in recoveryfrom injury to the liver, testis or thymic cells, compositionscontaining FGF-21 can be administered and it would likely be desirableto achieve certain target levels of FGF-21 in sera or in any desiredtissue compartment. It would, therefore, be advantageous to be able tomonitor the levels of FGF-21 in a patient. Accordingly, the presentinvention also provides methods for detecting the presence of FGF-21 ina sample from a patient.

The term “detection” as used herein in the context of detecting thepresence of FGF-21 in a patient is intended to include determining theamount of FGF-21 or the ability to express an amount of FGF-21 in apatient, distinguishing FGF-21 from other growth factors, the estimationof prognosis in terms of probable outcome of a degenerative disease andprospect for recovery, monitoring the FGF-21 levels over a period oftime as a measure of status of the condition, and monitoring FGF-21levels for determining a preferred therapeutic regimen for the patient.

To detect the presence of FGF-21 in a patient, a sample is obtained fromthe patient. The sample can be a tissue biopsy sample or a sample ofblood, plasma, serum, CSF or the like. FGF-21 is expressed in livertissues, as discussed in Example 2. Samples for detecting FGF-21 can betaken from this tissue. When assessing the levels of FGF-21 in theliver, thymus or testis, a preferred sample is a sample taken from thesetissues or from veins draining these tissues.

In some instances it is desirable to determine whether the FGF-21 geneis intact in the patient or in a tissue or cell line within the patient.By an intact FGF-21 gene it is meant that there are no alterations inthe gene such as point mutations, deletions, insertions, chromosomalbreakage, chromosomal rearrangements and the like wherein suchalteration might alter production of FGF-21 or alter its biologicalactivity, stability or the like to lead to disease processes orsusceptibility to cellular degenerative conditions. Thus, in oneembodiment of the present invention a method is provided for detectingand characterizing any alterations in the FGF-21 gene. The methodcomprises providing an oligonucleotide that contains the FGF-21 cDNA,genomic DNA or a fragment thereof or a derivative thereof. By aderivative of an oligonucleotide, it is meant that the derivedoligonucleotide is substantially the same as the sequence from which itis derived in that the derived sequence has sufficient sequencecomplementarily to the sequence from which it is derived to hybridize tothe FGF-21 gene. The derived nucleotide sequence is not necessarilyphysically derived from the nucleotide sequence, but may be generated inany manner including for example, chemical synthesis or DNA replicationor reverse transcription or transcription.

Typically, patient genomic DNA is isolated from a cell sample from thepatient and digested with one or more restriction endonucleases such as,for example, TaqI and AluI. Using the Southern blot protocol, which iswell known in the art, this assay determines whether a patient or aparticular tissue in a patient has an intact FGF-21 gene or an FGF-21gene abnormality.

Hybridization to an FGF-21 gene would involve denaturing the chromosomalDNA to obtain a single-stranded DNA; contacting the single-stranded DNAwith a gene probe associated with the FGF-21 gene sequence; andidentifying the hybridized DNA-probe to detect chromosomal DNAcontaining at least a portion of a human FGF-21 gene.

The term “probe” as used herein refers to a structure comprised of apolynucleotide that forms a hybrid structure with a target sequence, dueto complementarity of probe sequence with a sequence in the targetregion. Oligomers suitable for use as probes may contain a minimum ofabout 8-12 contiguous nucleotides which are complementary to thetargeted sequence and preferably a minimum of about 20.

The FGF-21 gene probes of the present invention can be DNA or RNAoligonucleotides and can be made by any method known in the art such as,for example, excision, transcription or chemical synthesis. Probes maybe labeled with any detectable label known in the art such as, forexample, radioactive or fluorescent labels or enzymatic marker. Labelingof the probe can be accomplished by any method known in the art such asby PCR, random priming, end labeling, nick translation or the like. Oneskilled in the art will also recognize that other methods not employinga labeled probe can be used to determine the hybridization. Examples ofmethods that can be used for detecting hybridization include Southernblotting, fluorescence in situ hybridization, and single-strandconformation polymorphism with PCR amplification.

Hybridization is typically carried out at 25°-45° C., more preferably at32°-40° C. and more preferably at 37°-38° C. The time required forhybridization is from about 0.25 to about 96 hours, more preferably fromabout one to about 72 hours, and most preferably from about 4 to about24 hours.

FGF-21 gene abnormalities can also be detected by using the PCR methodand primers that flank or lie within the FGF-21 gene. The PCR method iswell known in the art. Briefly, this method is performed using twooligonucleotide primers which are capable of hybridizing to the nucleicacid sequences flanking a target sequence that lies within an FGF-21gene and amplifying the target sequence. The terms “oligonucleotideprimer” as used herein refers to a short strand of DNA or RNA ranging inlength from about 8 to about 30 bases. The upstream and downstreamprimers are typically from about 20 to about 30 base pairs in length andhybridize to the flanking regions for replication of the nucleotidesequence. The polymerization is catalyzed by a DNA-polymerase in thepresence of deoxynucleotide triphosphates or nucleotide analogs toproduce double-stranded DNA molecules. The double strands are thenseparated by any denaturing method including physical, chemical orenzymatic. Commonly, the method of physical denaturation is usedinvolving heating the nucleic acid, typically to temperatures from about80° C. to 105° C. for times ranging from about 1 to about 10 minutes.The process is repeated for the desired number of cycles.

The primers are selected to be substantially complementary to the strandof DNA being amplified. Therefore, the primers need not reflect theexact sequence of the template, but must be sufficiently complementaryto selectively hybridize with the strand being amplified.

After PCR amplification, the DNA sequence comprising FGF-21 or pre-proFGF-21 or a fragment thereof is then directly sequenced and analyzed bycomparison of the sequence with the sequences disclosed herein toidentify alterations which might change activity or expression levels orthe like.

In another embodiment, a method for detecting FGF-21 is provided basedupon an analysis of tissue expressing the FGF-21 gene. Certain tissuessuch as those identified below in Example 2 have been found to expressthe FGF-21 gene. The method comprises hybridizing a polynucleotide tomRNA from a sample of tissues that normally express the FGF-21 gene. Thesample is obtained from a patient suspected of having an abnormality inthe FGF-21 gene or in the FGF-21 gene of particular cells.

To detect the presence of mRNA encoding FGF-21 protein, a sample isobtained from a patient. The sample can be from blood or from a tissuebiopsy sample. The sample may be treated to extract the nucleic acidscontained therein. The resulting nucleic acid from the sample issubjected to gel electrophoresis or other size separation techniques.

The mRNA of the sample is contacted with a DNA sequence serving as aprobe to form hybrid duplexes. The use of a labeled probes as discussedabove allows detection of the resulting duplex.

When using the cDNA encoding FGF-21 protein or a derivative of the cDNAas a probe, high stringency conditions can be used in order to preventfalse positives, that is the hybridization and apparent detection ofFGF-21 nucleotide sequences when in fact an intact and functioningFGF-21 gene is not present. When using sequences derived from the FGF-21cDNA, less stringent conditions could be used, however, this would be aless preferred approach because of the likelihood of false positives.The stringency of hybridization is determined by a number of factorsduring hybridization and during the washing procedure, includingtemperature, ionic strength, length of time and concentration offormamide. These factors are outlined in, for example, Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2^(nd) Ed. (1989) Cold SpringHarbor Press, Cold Spring Harbor, N.Y.

In order to increase the sensitivity of the detection in a sample ofmRNA encoding the FGF-21 protein, the technique of reversetranscription/polymerization chain reaction (RT/PCR) can be used toamplify cDNA transcribed from mRNA encoding the FGF-21 protein. Themethod of RT/PCR is well known in the art, and can be performed asfollows. Total cellular RNA is isolated by, for example, the standardguanidium isothiocyanate method and the total RNA is reversetranscribed. The reverse transcription method involves synthesis of DNAon a template of RNA using a reverse transcriptase enzyme and a 3′ endprimer. Typically, the primer contains an oligo(dT) sequence. The cDNAthus produced is then amplified using the PCR method and FGF-21 specificprimers. (Belyavsky et al., Nucl. Acid Res. 17:2919-2932, 1989; Krug andBerger, Methods in Enzymology, 152:316-325, Academic Press, NY, 1987which are incorporated by reference).

The polymerase chain reaction method is performed as described aboveusing two oligonucleotide primers that are substantially complementaryto the two flanking regions of the DNA segment to be amplified.

Following amplification, the PCR product is then electrophoresed anddetected by ethidium bromide staining or by phosphoimaging.

The present invention further provides for methods to detect thepresence of the FGF-21 protein in a sample obtained from a patient. Anymethod known in the art for detecting proteins can be used. Such methodsinclude, but are not limited to immunodiffusion, immunoelectrophoresis,immunochemical methods, binder-ligand assays, immunohistochemicaltechniques, agglutination and complement assays. (for example, see Basicand Clinical Immunology, 217-262, Sites and Terr, eds., Appleton &Lange, Norwalk, Conn., 1991 which is incorporated by reference).Preferred are binder-ligand immunoassay methods including reactingantibodies with an epitope or epitopes of the FGF-21 protein andcompetitively displacing a labeled FGF-21 protein or derivative thereof.Preferred antibodies are prepared according to Example 4.

As used herein, a derivative of the FGF-21 protein is intended toinclude a polypeptide in which certain amino acids have been deleted orreplaced or changed to modified or unusual amino acids wherein theFGF-21 derivative is biologically equivalent to FGF-21 and wherein thepolypeptide derivative cross-reacts with antibodies raised against theFGF-21 protein. By cross-reaction it is meant that an antibody reactswith an antigen other than the one that induced its formation.

Numerous competitive and non-competitive protein binding immunoassaysare well known in the art. Antibodies employed in such assays may beunlabeled, for example as used in agglutination tests, or labeled foruse in a wide variety of assay methods. Labels that can be used includeradionuclides, enzymes, fluorescers, chemiluminescers, enzyme substratesor co-factors, enzyme inhibitors, particles, dyes and the like for usein radioimmunoassay (RIA), enzyme immunoassays, e.g., enzyme-linkedimmunosorbent assay (ELISA), fluorescent immunoassays and the like.

Polyclonal or monoclonal antibodies to the FGF-21 protein or an epitopethereof can be made for use in immunoassays by any of a number ofmethods known in the art. By epitope reference is made to an antigenicdeterminant of a polypeptide. An epitope could comprise 3 amino acids ina spatial conformation which is unique to the epitope. Generally anepitope consists of at least 5 such amino acids. Methods of determiningthe spatial conformation of amino acids are known in the art, andinclude, for example, x-ray crystallography and 2 dimensional nuclearmagnetic resonance.

One approach for preparing antibodies to a protein is the selection andpreparation of an amino acid sequence of all or part of the protein,chemically synthesizing the sequence and injecting it into anappropriate animal, usually a rabbit or a mouse (see Example 4)

Oligopeptides can be selected as candidates for the production of anantibody to the FGF-21 protein based upon the oligopeptides lying inhydrophilic regions, which are thus likely to be exposed in the matureprotein. Preferred oligopeptides are RQRYLYTDDAQQREAH (SEQ ID NO:7)(residues 46-61 of SEQ NO:4) and HLPGNKSPHRDPAPR (SEQ ID NO:8) (residues146-160 of SEQ ID NO:4). Additional oligopeptides can be determinedusing, for example, the Antigenicity Index of Welling, G.W. et al., FEBSLett. 188:215-218, 1985, incorporated herein by reference.

Antibodies to FGF-21 can also be raised against oligopeptides thatinclude one or more of the conserved regions identified herein such thatthe antibody can cross-react with other family members. Such antibodiescan be used to identify and isolate the other family members.

Methods for preparation of the FGF-21 protein or an epitope thereofinclude, but are not limited to chemical synthesis, recombinant DNAtechniques or isolation from biological samples. Chemical synthesis of apeptide can be performed, for example, by the classical Merrifeld methodof solid phase peptide synthesis (Merrifeld, J. Am. Chem. Soc. 85:2149,1963 which is incorporated by reference) or the FMOC strategy on a RapidAutomated Multiple Peptide Synthesis system (E. I. du Pont de NemoursCompany, Wilmington, Del.) (Caprino and Han, J. Org. Chem. 37:3404, 1972which is incorporated by reference).

Polyclonal antibodies can be prepared by immunizing rabbits or otheranimals by injecting antigen followed by subsequent boosts atappropriate intervals. The animals are bled and sera assayed againstpurified FGF-21 protein usually by ELISA or by bioassay based upon theability to block the action of FGF-21 on liver or other cells. Whenusing avian species, e.g., chicken, turkey and the like, the antibodycan be isolated from the yolk of the egg. Monoclonal antibodies can beprepared after the method of Milstein and Kohler by fusing splenocytesfrom immunized mice with continuously replicating tumor cells such asmyeloma or lymphoma cells. (Milstein and Kohler, Nature 256:495-497,1975; Gulfre and Milstein, Methods in Enzymology: ImmunochemicalTechniques 73:1-46, Langone and Banatis eds., Academic Press, 1981 whichare incorporated by reference). The hybridoma cells so formed are thencloned by limiting dilution methods and supernates assayed for antibodyproduction by ELISA, RIA or bioassay.

The unique ability of antibodies to recognize and specifically bind totarget proteins provides an approach for treating an overexpression ofthe protein. Thus, another aspect of the present invention provides fora method for preventing or treating diseases involving overexpression ofthe FGF-21 protein by treatment of a patient with specific antibodies tothe FGF-21 protein.

Specific antibodies, either polyclonal or monoclonal, to the FGF-21protein can be produced by any suitable method known in the art asdiscussed above. For example, murine or human monoclonal antibodies canbe produced by hybridoma technology or, alternatively, the FGF-21protein, or an immunologically active fragment thereof, or ananti-idiotypic antibody, or fragment thereof can be administered to ananimal to elicit the production of antibodies capable of recognizing andbinding to the FGF-21 protein. Such antibodies can be from any class ofantibodies including, but not limited to IgG, IgA, IgM, IgD, and IgE orin the case of avian species, IgY and from any subclass of antibodies.

Polypeptides encoded by the instant polynucleotides and correspondingfull-length genes can be used to screen peptide libraries, proteinlibraries, small molecule libraries, and phage display libraries, andother known methods, to identify analogs or antagonists.

Native FGF polypeptides may play a role in cancer. For example, FGFfamily members can induce marked morphological transformation of NIH 3T3cells, and exhibit strong tumorigenicity in nude mice. Angiogenicactivity has been exhibited by FGF family members. Thus, inhibitors ofFGF can be used to treat cancer, such as prostate cancer.

A library of peptides may be synthesized following the methods disclosedin U.S. Pat. No. 5,010,175, and in PCT No. WO 91/17823. As describedbelow in brief, a mixture of peptides is prepared, which is thenscreened to identify the peptides exhibiting the desired signaltransduction and receptor binding activity. According to the method ofthe ′175 patent, a suitable peptide synthesis support (e.g., a resin) iscoupled to a mixture of appropriately protected, activated amino acids.The concentration of each amino acid in the reaction mixture is balancedor adjusted in inverse proportion to its coupling reaction rate so thatthe product is an equimolar mixture of amino acids coupled to thestarting resin. The bound amino acids are then deprotected, and reactedwith another balanced amino acid mixture to form an equimolar mixture ofall possible dipeptides. This process is repeated until a mixture ofpeptides of the desired length (e.g., hexamers) is formed. Note that oneneed not include all amino acids in each step: one may include only oneor two amino acids in some steps (e.g., where it is known that aparticular amino acid is essential in a given position), thus reducingthe complexity of the mixture. After the synthesis of the peptidelibrary is completed, the mixture of peptides is screened for binding tothe selected polypeptide. The peptides are then tested for their abilityto inhibit or enhance activity. Peptides exhibiting the desired activityare then isolated and sequenced.

The method described in PCT No. WO 91/17823 is similar. However, insteadof reacting the synthesis resin with a mixture of activated amino acids,the resin is divided into twenty equal portions (or into a number ofportions corresponding to the number of different amino acids to beadded in that step), and each amino acid is coupled individually to itsportion of resin. The resin portions are then combined, mixed, and againdivided into a number of equal portions for reaction with the secondamino acid. In this manner, each reaction may be easily driven tocompletion. Additionally, one may maintain separate “subpools” bytreating portions in parallel, rather than combining all resins at eachstep. This simplifies the process of determining which peptides areresponsible for any observed receptor binding or signal transductionactivity.

In such cases, the subpools containing, e.g., 1-2,000 candidates eachare exposed to one or more polypeptides of the invention. Each subpoolthat produces a positive result is then resynthesized as a group ofsmaller subpools (sub-subpools) containing, e.g., 20-100 candidates, andreassayed. Positive sub-subpools may be resynthesized as individualcompounds, and assayed finally to determine the peptides that exhibit ahigh binding constant. These peptides can be tested for their ability toinhibit or enhance the native activity. The methods described in PCT No.WO 91/7823 and U.S. Pat. No. 5,194,392 (herein incorporated byreference) enable the preparation of such pools and subpools byautomated techniques in parallel, such that all synthesis andresynthesis may be performed in a matter of days.

Peptide agonists or antagonists are screened using any available method,such as signal transduction, antibody binding, receptor binding andmitogenic assays. The assay conditions ideally should resemble theconditions under which the native activity is exhibited in vivo, thatis, under physiologic pH, temperature, and ionic strength. Suitableagonists or antagonists will exhibit strong inhibition or enhancement ofthe native activity at concentrations that do not cause toxic sideeffects in the subject. Agonists or antagonists that compete for bindingto the native polypeptide may require concentrations equal to or greaterthan the native concentration, while inhibitors capable of bindingirreversibly to the polypeptide may be added in concentrations on theorder of the native concentration.

The availability of hFGF-21 and mFGF-21 allows for the identification ofsmall molecules and low molecular weight compounds that inhibit thebinding of FGF-21 to its receptor, through routine application ofhigh-throughput screening methods (HTS). HTS methods generally refer totechnologies that permit the rapid assaying of lead compounds fortherapeutic potential. HTS techniques employ robotic handling of testmaterials, detection of positive signals, and interpretation of data.Lead compounds may be identified via the incorporation of radioactivityor through optical assays that rely on absorbance, fluorescence orluminescence as read-outs. Gonzalez, J. E. et al., (1998) Curr. Opin.Biotech. 9:624-631. Assays for detecting interaction between an FGFmolecule and FGF receptor are described in, for example, Blunt, A. G. etal., (1997) J. Biol. Chem. 272:3733-3738, and such assays can be adaptedfor determining if a candidate molecule can inhibit the interactionbetween FGF-21 and its receptor.

Model systems are available that can be adapted for use in highthroughput screening for compounds that inhibit the interaction ofFGF-21 with its receptor, for example by competing with FGF-21 forreceptor binding. Sarubbi et al., (1996) Anal Biochem. 237:70-75describe cell-free, non-isotopic assays for identifying molecules thatcompete with natural ligands for binding to the active site of IL-1receptor. Martens, C. et al., (1999) Anal. Biochem. 273:20-31 describe ageneric particle-based nonradioactive method in which a labeled ligandbinds to its receptor immobilized on a particle; label on the particledecreases in the presence of a molecule that competes with the labeledligand for receptor binding.

The therapeutic FGF-21 polynucleotides and polypeptides of the presentinvention may be utilized in gene delivery vehicles. The gene deliveryvehicle may be of viral or non-viral origin (see generally, Jolly,Cancer Gene Therapy 1:51-64 (1994); Kimura, Human Gene Therapy 5:845-852(1994); Connelly, Human Gene Therapy 1:185-193 (1995); and Kaplitt,Nature Genetics 6:148-153 (1994)). Gene therapy vehicles for delivery ofconstructs including a coding sequence of a therapeutic of the inventioncan be administered either locally or systemically. These constructs canutilize viral or non-viral vector approaches. Expression of such codingsequences can be induced using endogenous mammalian or heterologouspromoters. Expression of the coding sequence can be either constitutiveor regulated.

The present invention can employ recombinant retroviruses which areconstructed to carry or express a selected nucleic acid molecule ofinterest. Retrovirus vectors that can be employed include thosedescribed in EP 0 415 731; WO 90/07936; WO 94/03622; WO 93/25698; WO93/25234; U.S. Pat. No. 5,219,740; WO 93/11230; WO 93/10218; Vile andHart, Cancer Res. 53:3860-3864 (1993); Vile and Hart, Cancer Res.53:962-967 (1993); Ram et al., Cancer Res. 53:83-88 (1993); Takamiya etal, J. Neurosci. Res. 33:493-503 (1992); Baba et al., J. Neurosurg.79:729-735 (1993); U.S. Pat. No. 4,777,127; GB Patent No. 2,200,651; andEP 0 345 242. Preferred recombinant retroviruses include those describedin WO 91/02805.

Packaging cell lines suitable for use with the above-describedretroviral vector constructs may be readily prepared (see PCTpublications WO 95/30763 and WO 92/05266), and used to create producercell lines (also termed vector cell lines) for the production ofrecombinant vector particles. Within particularly preferred embodimentsof the invention, packaging cell lines are made from human (such asHT1080 cells) or mink parent cell lines, thereby allowing production ofrecombinant retroviruses that can survive inactivation in human serum.

The present invention also employs alphavirus-based vectors that canfunction as gene delivery vehicles. Such vectors can be constructed froma wide variety of alphaviruses, including, for example, Sindbis virusvectors, Semliki forest virus (ATCC VR-67; ATCC VR-1247), Ross Rivervirus (ATCC VR-373; ATCC VR-1246) and Venezuelan equine encephalitisvirus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532).Representative examples of such vector systems include those describedin U.S. Pat. Nos. 5,091,309; 5,217,879; and 5,185,440; and PCTPublication Nos. WO 92/10578; WO 94/21792; WO 95/27069; WO 95/27044; andWO 95/07994.

Gene delivery vehicles of the present invention can also employparvovirus such as adeno-associated virus (AAV) vectors. Representativeexamples include the AAV vectors disclosed by Srivastava in WO 93/09239,Samulski et al., J. Vir. 63:3822-3828 (1989); Mendelson et al., Virol.166:154-165 (1988); and Flotte et al., P.N.A.S. 90:10613-10617 (1993).

Representative examples of adenoviral vectors include those described byBerkner, Biotechniques 6:616-627 (Biotechniques); Rosenfeld et al.,Science 252:431-434 (1991); WO 93/19191; Kolls et al., P.N.A.S. :215-219(1994); Kass-Eisler et al., P.N.A.S. 90:11498-11502 (1993); Guzman etal., Circulation 88:2838-2848 (1993); Guzman et al., Cir. Res.73:1202-1207 (1993); Zabner et al., Cell 75:207-216 (1993); Li et al.,Hum. Gene Ther. 4:403-409 (1993); Cailaud et al., Eur. J. Neurosci.5:1287-1291 (1993); Vincent et al., Nat. Genet. 5:130-134 (1993); Jaffeet al., Nat. Genet. 1:372-378 (1992); and Levrero et al., Gene101:195-202 (1992). Exemplary adenoviral gene therapy vectors employablein this invention also include those described in WO 94/12649, WO93/03769; WO 93/19191; WO 94/28938; WO95/11984 and WO 95/00655.Administration of DNA linked to killed adenovirus as described inCuriel, Hum. Gene Ther. 3:147-154 (1992) may be employed.

Other gene delivery vehicles and methods may be employed, includingpolycationic condensed DNA linked or unlinked to killed adenovirusalone, for example Curiel, Hum. Gene Ther. 3:147-154 (1992);ligand-linked DNA, for example see Wu, J. Biol. Chem. 264:16985-16987(1989); eukaryotic cell delivery vehicles cells, for example see U.S.Ser. No. 08/240,030, filed May 9, 1994, and U.S. Ser. No. 08/404,796;

deposition of photopolymerized hydrogel materials; hand-held genetransfer particle gun, as described in U.S. Pat. No. 5,149,655; ionizingradiation as described in U.S. Pat. No. 5,206,152 and in WO 92/11033;nucleic charge neutralization or fusion with cell membranes. Additionalapproaches are described in Philip, Mol. Cell Biol. 14:2411-2418 (1994),and in Woffendin, Proc. Natl. Acad Sci. 91:1581-1585 (1994).

Naked DNA may also be employed. Exemplary naked DNA introduction methodsare described in WO 90/11092 and U.S. Pat. No. 5,580,859. Uptakeefficiency may be improved using biodegradable latex beads. DNA coatedlatex beads are efficiently transported into cells after endocytosisinitiation by the beads. The method may be improved further by treatmentof the beads to increase hydrophobicity and thereby facilitatedisruption of the endosome and release of the DNA into the cytoplasm.Liposomes that can act as gene delivery vehicles are described in U.S.Pat. No. 5,422,120, PCT Patent Publication Nos. WO 95/13796, WO94/23697, and WO 91/14445, and EP No. 0 524 968.

Further non-viral delivery suitable for use includes mechanical deliverysystems such as the approach described in Woffendin et al., Proc. Natl.Acad Sci. USA 91(24):11581-11585 (1994). Moreover, the coding sequenceand the product of expression of such can be delivered throughdeposition of photopolymerized hydrogel materials. Other conventionalmethods for gene delivery that can be used for delivery of the codingsequence include, for example, use of hand-held gene transfer particlegun, as described in U.S. Pat. No. 5,149,655; use of ionizing radiationfor activating transferred gene, as described in U.S. Pat. No.5,206,152and PCT Patent Publication No. WO 92/11033.

FGF has been implicated in diseases characterized by loss of function,inadequate function/number, abnormal function or death of cells, tissuesor organs for which function or survival can be prolonged/rescued, andabnormalities reversed or prevented by therapy with FGF.

Loss of pulmonary, bronchia or alveolar cells or function, healing ofpulmonary or bronchia wounds, pulmonary infraction, emphysema/chronicobstructive pulmonary disease, asthma, sequelae of infectious orautoimmune disease, sequelae of pulmonary arterial or venoushypertension, pulmonary fibrosis, pulmonary disease of immaturity, andcystic fibrosis are conditions amenable to treatment with FGF.

Ischemic vascular disease may be amenable to FGF-21 treatment, whereinthe disease is characterized by inadequate blood flow to an organ(s).Treatment may induce therapeutic angiogenesis or preservefunction/survival of cells (myocardial ischemia/infarction, peripheralvascular disease, renal artery disease, stroke). Cardiomyopathiescharacterized by loss of function or death of cardiac myocytes orsupporting cells in the heart (congestive heart failure, myocarditis)may also be treated using FGF-21, as can musculoskeletal diseasecharacterized by loss of function, inadequate function or death ofskeletal muscle cells, bone cells or supporting cells. Examples includeskeletal myopathies, bone disease, and arthritis.

FGF-21 polynucleotides and polypeptides may aid in correction ofcongenital defects due to loss of FGF-21 molecule or its function(liver, heart, lung, brain, limbs, kidney, etc.).

Treatment of wound healing is yet another use of FGF-21 polypeptides andpolynucleotides, either due to trauma, disease, medical or surgicaltreatment, including regeneration of cell populations and tissuesdepleted by these processes. Examples include liver regeneration,operative wound healing, re-endothelialization of injured blood vessels,healing of traumatic wounds, healing of ulcers due to vascular,metabolic disease, etc., bone fractures, loss of cells due toinflammatory disease, etc.

FGF-21 may also be used in screens to identify drugs for treatment ofcancers which involve over activity of the molecule, or new targetswhich would be useful in the identification of new drugs.

For all of the preceding embodiments, the clinician will determine,based on the specific condition, whether FGF-21 polypeptides orpolynucleotides, antibodies to FGF-21, or small molecules such aspeptide analogues or antagonists, will be the most suitable form oftreatment. These forms are all within the scope of the invention.

Preferred embodiments of the invention are described in the followingexamples. Other embodiments within the scope of the claims herein willbe apparent to one skilled in the art from consideration of thespecification or practice of the invention as disclosed herein. It isintended that the specification, together with the examples, beconsidered exemplary only, with the scope and spirit of the inventionbeing indicated by the claims which follow the examples.

EXAMPLES Example 1

Isolation and Analysis of Mouse FGF-21

DNA was prepared from mouse embryo cDNA. DNA was amplified from mouseembryo cDNA by polymerase chain reaction (PCR) for 30 cycles in 25 μl ofa reaction mixture containing each of the sense and antisense degenerateprimers representing all possible codons corresponding to the amino acidsequences of human FGF-19, RPYDGYN (SEQ ID NO: 14) and LPMLPM (SEQ IDNO: 15), respectively. The amplified product was further amplified byPCR with each of the sense and antisense degenerate primers representingall possible codons corresponding to the amino acid sequences of humanFGF-19, RPDGYN (SEQ ID NO: 16) and HFLPML (SEQ ID NO: 17), respectively.The amplified DNAs of expected size (approximately 120 base pairs) werecloned. By determination of the nucleotide sequences of the cloned DNAs,a novel mouse FGF, FGF-21, cDNA was identified. To determine the entirecoding region of the novel FGF cDNA, the coding region was amplifiedfrom mouse embryo cDNA by adaptor-ligation mediated PCR using a MarathoncDNA amplification kit (Clontech, Palo Alto, California) and primersspecific for the FGF. The cDNA encoding the entire coding region of theFGF was amplified from mouse embryo cDNA by PCR using the FGF-specificprimers including the 5′ and 3′ noncoding sequences, and cloned into thepGEM-T DNA vector. The nucleotide sequence is shown in SEQ ID NO: 1 andthe amino acid sequence is shown in SEQ ID NO:2.

Example 2

Expression of FGF-21 in Mouse Tissues

Poly (A)⁺RNA (10 μg) from mouse tissues was dissolved on a denaturingagarose gel (1%) containing formaldehyde, and transferred to anitrocellulose membrane in 20×SSC (1×SSC:0.15 M NACl/0.015 M sodiumcitrate) overnight. A ³²P-labeled FGF-21 cDNA probe (˜650 base pairs)was labeled with a random primer labeling kit (Pharmacia Biotech,Uppsala, Sweden) and deoxycytidine 5′-[α-³²P-] triphosphate (˜110TBq/mmol) (ICN Biomedicals Inc., Costa Mesa, Calif.). The membrane wasincubated in hybridization solution containing the labeled probe asdescribed (Hoshikawa et al., Biochem. Biophys. Res. Commun. 244:187-191(1998)), and analyzed with a radio-imaging analyzer (BAS 2000, FujiPhoto Film Co., Tokyo, Japan). As shown in FIG. 3, FGF-21 expression wasmost predominant in liver, with expression also seen in testis andthymus.

Example 3

Isolation and Analysis of Human FGF-21

The human FGF-21 gene was located in the 5′ flanking region of aputative human alpha 1,2-fucosyltransferase gene. The cDNA encoding theentire coding region of human FGF-21 was amplified from fetal brain cDNAby PCR using the FGF-specific primers including the 5′ and 3′ noncodingsequences, and cloned into the pGEM-T DNA vector. The protein contains209 amino acids, as shown in SEQ ID NO:4 (FIG. 5), and is encoded by thepolynucleotide sequence of SEQ ID NO:3. Primers for amplification ofhuman FGF-21 cDNA coding region are: sense primer for FGF-21: 5′agccattgatggactcggac3′; antisense primer for FGF-21: 5′tggcttcaggaagcgtagct 3′.

Example 4

Preparation of Antisera to FGF-21 by Immunization of Rabbits with anFGF-21 Peptide

A peptide sequence corresponding to selected contiguous amino acids ofthe human FGF-21 protein is synthesized and coupled to keyhole limpethemocyanin (KLH) as described (Harlow and Land, Antibodies: A LaboratoryManual, 1988. Cold Spring Harbor Laboratory, New York, N.Y.) TheKLH-coupled peptide is used to immunize rabbits. Antisera are tested forspecificity to FGF-21, and for cross-reactivity with other FGF proteins.

Exemplary peptide sequences are:

1. RQRYLYDDAQQTEAH (residues 46-61 of SEQ ID NO:4)

2. HLPGNKSPHRDPAPR (residues 146-160 of SEQ ID NO:4)

All patents, published patent applications and publications cited hereinare incorporated by reference as if set forth fully herein.

Although certain preferred embodiments have been described herein, it isnot intended that such embodiments be construed as limitations on thescope of the invention except as set forth in the following claims.

17 1 659 DNA Mus musculus CDS (14)...(646) 1 gagcgcagcc ctg atg gaa tggatg aga tct aga gtt ggg acc ctg gga 49 Met Glu Trp Met Arg Ser Arg ValGly Thr Leu Gly 1 5 10 ctg tgg gtc cga ctg ctg ctg gct gtc ttc ctg ctgggg gtc tac caa 97 Leu Trp Val Arg Leu Leu Leu Ala Val Phe Leu Leu GlyVal Tyr Gln 15 20 25 gca tac ccc atc cct gac tcc agc ccc ctc ctc cag tttggg ggt caa 145 Ala Tyr Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe GlyGly Gln 30 35 40 gtc cgg cag agg tac ctc tac aca gat gac gac caa gac actgaa gcc 193 Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Asp Gln Asp Thr GluAla 45 50 55 60 cac ctg gag atc agg gag gat gga aca gtg gta ggc gca gcacac cgc 241 His Leu Glu Ile Arg Glu Asp Gly Thr Val Val Gly Ala Ala HisArg 65 70 75 agt cca gaa agt ctc ctg gag ctc aaa gcc ttg aag cca ggg gtcatt 289 Ser Pro Glu Ser Leu Leu Glu Leu Lys Ala Leu Lys Pro Gly Val Ile80 85 90 caa atc ctg ggt gtc aaa gcc tct agg ttt ctt tgc caa cag cca gat337 Gln Ile Leu Gly Val Lys Ala Ser Arg Phe Leu Cys Gln Gln Pro Asp 95100 105 gga gct ctc tat gga tcg cct cac ttt gat cct gag gcc tgc agc ttc385 Gly Ala Leu Tyr Gly Ser Pro His Phe Asp Pro Glu Ala Cys Ser Phe 110115 120 aga gaa ctg ctg ctg gag gac ggt tac aat gtg tac cag tct gaa gcc433 Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala 125130 135 140 cat ggc ctg ccc ctg cgt ctg cct cag aag gac tcc cca aac caggat 481 His Gly Leu Pro Leu Arg Leu Pro Gln Lys Asp Ser Pro Asn Gln Asp145 150 155 gca aca tcc tgg gga cct gtg cgc ttc ctg ccc atg cca ggc ctgctc 529 Ala Thr Ser Trp Gly Pro Val Arg Phe Leu Pro Met Pro Gly Leu Leu160 165 170 cac gag ccc caa gac caa gca gga ttc ctg ccc cca gag ccc ccagat 577 His Glu Pro Gln Asp Gln Ala Gly Phe Leu Pro Pro Glu Pro Pro Asp175 180 185 gtg ggc tcc tct gac ccc ctg agc atg gta gag cct tta cag ggccga 625 Val Gly Ser Ser Asp Pro Leu Ser Met Val Glu Pro Leu Gln Gly Arg190 195 200 agc ccc agc tat gcg tcc tga ctcttcctga atc 659 Ser Pro SerTyr Ala Ser * 205 210 2 210 PRT Mus musculus 2 Met Glu Trp Met Arg SerArg Val Gly Thr Leu Gly Leu Trp Val Arg 1 5 10 15 Leu Leu Leu Ala ValPhe Leu Leu Gly Val Tyr Gln Ala Tyr Pro Ile 20 25 30 Pro Asp Ser Ser ProLeu Leu Gln Phe Gly Gly Gln Val Arg Gln Arg 35 40 45 Tyr Leu Tyr Thr AspAsp Asp Gln Asp Thr Glu Ala His Leu Glu Ile 50 55 60 Arg Glu Asp Gly ThrVal Val Gly Ala Ala His Arg Ser Pro Glu Ser 65 70 75 80 Leu Leu Glu LeuLys Ala Leu Lys Pro Gly Val Ile Gln Ile Leu Gly 85 90 95 Val Lys Ala SerArg Phe Leu Cys Gln Gln Pro Asp Gly Ala Leu Tyr 100 105 110 Gly Ser ProHis Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu 115 120 125 Leu GluAsp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu Pro 130 135 140 LeuArg Leu Pro Gln Lys Asp Ser Pro Asn Gln Asp Ala Thr Ser Trp 145 150 155160 Gly Pro Val Arg Phe Leu Pro Met Pro Gly Leu Leu His Glu Pro Gln 165170 175 Asp Gln Ala Gly Phe Leu Pro Pro Glu Pro Pro Asp Val Gly Ser Ser180 185 190 Asp Pro Leu Ser Met Val Glu Pro Leu Gln Gly Arg Ser Pro SerTyr 195 200 205 Ala Ser 210 3 643 DNA Homo sapiens CDS (9)...(638) 3agccattg atg gac tcg gac gag acc ggg ttc gag cac tca gga ctg tgg 50 MetAsp Ser Asp Glu Thr Gly Phe Glu His Ser Gly Leu Trp 1 5 10 gtt tct gtgctg gct ggt ctt ctg ctg gga gcc tgc cag gca cac ccc 98 Val Ser Val LeuAla Gly Leu Leu Leu Gly Ala Cys Gln Ala His Pro 15 20 25 30 atc cct gactcc agt cct ctc ctg caa ttc ggg ggc caa gtc cgg cag 146 Ile Pro Asp SerSer Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Gln 35 40 45 cgg tac ctc tacaca gat gat gcc cag cag aca gaa gcc cac ctg gag 194 Arg Tyr Leu Tyr ThrAsp Asp Ala Gln Gln Thr Glu Ala His Leu Glu 50 55 60 atc agg gag gat gggacg gtg ggg ggc gct gct gac cag agc ccc gaa 242 Ile Arg Glu Asp Gly ThrVal Gly Gly Ala Ala Asp Gln Ser Pro Glu 65 70 75 agt ctc ctg cag ctg aaagcc ttg aag ccg gga gtt att caa atc ttg 290 Ser Leu Leu Gln Leu Lys AlaLeu Lys Pro Gly Val Ile Gln Ile Leu 80 85 90 gga gtc aag aca tcc agg ttcctg tgc cag cgg cca gat ggg gcc ctg 338 Gly Val Lys Thr Ser Arg Phe LeuCys Gln Arg Pro Asp Gly Ala Leu 95 100 105 110 tat gga tcg ctc cac tttgac cct gag gcc tgc agc ttc cgg gag ctg 386 Tyr Gly Ser Leu His Phe AspPro Glu Ala Cys Ser Phe Arg Glu Leu 115 120 125 ctt ctt gag gac gga tacaat gtt tac cag tcc gaa gcc cac ggc ctc 434 Leu Leu Glu Asp Gly Tyr AsnVal Tyr Gln Ser Glu Ala His Gly Leu 130 135 140 ccg ctg cac ctg cca gggaac aag tcc cca cac cgg gac cct gca ccc 482 Pro Leu His Leu Pro Gly AsnLys Ser Pro His Arg Asp Pro Ala Pro 145 150 155 cga gga cca gct cgc ttcctg cca cta cca ggc ctg ccc ccc gca ctc 530 Arg Gly Pro Ala Arg Phe LeuPro Leu Pro Gly Leu Pro Pro Ala Leu 160 165 170 ccg gag cca ccc gga atcctg gcc ccc cag ccc ccc gat gtg ggc tcc 578 Pro Glu Pro Pro Gly Ile LeuAla Pro Gln Pro Pro Asp Val Gly Ser 175 180 185 190 tcg gac cct ctg agcatg gtg gga cct tcc cag ggc cga agc ccc agc 626 Ser Asp Pro Leu Ser MetVal Gly Pro Ser Gln Gly Arg Ser Pro Ser 195 200 205 tac gct tcc tgaagcca 643 Tyr Ala Ser * 4 209 PRT Homo sapiens 4 Met Asp Ser Asp Glu ThrGly Phe Glu His Ser Gly Leu Trp Val Ser 1 5 10 15 Val Leu Ala Gly LeuLeu Leu Gly Ala Cys Gln Ala His Pro Ile Pro 20 25 30 Asp Ser Ser Pro LeuLeu Gln Phe Gly Gly Gln Val Arg Gln Arg Tyr 35 40 45 Leu Tyr Thr Asp AspAla Gln Gln Thr Glu Ala His Leu Glu Ile Arg 50 55 60 Glu Asp Gly Thr ValGly Gly Ala Ala Asp Gln Ser Pro Glu Ser Leu 65 70 75 80 Leu Gln Leu LysAla Leu Lys Pro Gly Val Ile Gln Ile Leu Gly Val 85 90 95 Lys Thr Ser ArgPhe Leu Cys Gln Arg Pro Asp Gly Ala Leu Tyr Gly 100 105 110 Ser Leu HisPhe Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu Leu 115 120 125 Glu AspGly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu Pro Leu 130 135 140 HisLeu Pro Gly Asn Lys Ser Pro His Arg Asp Pro Ala Pro Arg Gly 145 150 155160 Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala Leu Pro Glu 165170 175 Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val Gly Ser Ser Asp180 185 190 Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg Ser Pro Ser TyrAla 195 200 205 Ser 5 20 DNA Artificial Sequence PCR primer 5 agccattgatggactcggac 20 6 20 DNA Artificial Sequence PCR primer 6 tggcttcaggaagcgtagct 20 7 16 PRT Homo sapiens 7 Arg Gln Arg Tyr Leu Tyr Thr AspAsp Ala Gln Gln Thr Glu Ala His 1 5 10 15 8 15 PRT Homo sapiens 8 HisLeu Pro Gly Asn Lys Ser Pro His Arg Asp Pro Ala Pro Arg 1 5 10 15 9 218PRT Mus musculus 9 Met Ala Arg Lys Trp Asn Gly Arg Ala Val Ala Arg AlaLeu Val Leu 1 5 10 15 Ala Thr Leu Trp Leu Ala Val Ser Gly Arg Pro LeuAla Gln Gln Ser 20 25 30 Gln Ser Val Ser Asp Glu Asp Pro Leu Phe Leu TyrGly Trp Gly Lys 35 40 45 Ile Thr Arg Leu Gln Tyr Leu Tyr Ser Ala Gly ProTyr Val Ser Asn 50 55 60 Cys Phe Leu Arg Ile Arg Ser Asp Gly Ser Val AspCys Glu Glu Asp 65 70 75 80 Gln Asn Glu Arg Asn Leu Leu Glu Phe Arg AlaVal Ala Leu Lys Thr 85 90 95 Ile Ala Ile Lys Asp Val Ser Ser Val Arg TyrLeu Cys Met Ser Ala 100 105 110 Asp Gly Lys Ile Tyr Gly Leu Ile Arg TyrSer Glu Glu Asp Cys Thr 115 120 125 Phe Arg Glu Glu Met Asp Cys Leu GlyTyr Asn Gln Tyr Arg Ser Met 130 135 140 Lys His His Leu His Ile Ile PheIle Gln Ala Lys Pro Arg Glu Gln 145 150 155 160 Leu Gln Asp Gln Lys ProSer Asn Phe Ile Pro Val Phe His Arg Ser 165 170 175 Phe Phe Glu Thr GlyAsp Gln Leu Arg Ser Lys Met Phe Ser Leu Pro 180 185 190 Leu Glu Ser AspSer Met Asp Pro Phe Arg Met Val Glu Asp Val Asp 195 200 205 His Leu ValLys Ser Pro Ser Phe Gln Lys 210 215 10 216 PRT Homo sapiens 10 Met ArgSer Gly Cys Val Val Val His Val Trp Ile Leu Ala Gly Leu 1 5 10 15 TrpLeu Ala Val Ala Gly Arg Pro Leu Ala Phe Ser Asp Ala Gly Pro 20 25 30 HisVal His Tyr Gly Trp Gly Asp Pro Ile Arg Leu Arg His Leu Tyr 35 40 45 ThrSer Gly Pro His Gly Leu Ser Ser Cys Phe Leu Arg Ile Arg Ala 50 55 60 AspGly Val Val Asp Cys Ala Arg Gly Gln Ser Ala His Ser Leu Leu 65 70 75 80Glu Ile Lys Ala Val Ala Leu Arg Thr Val Ala Ile Lys Gly Val His 85 90 95Ser Val Arg Tyr Leu Cys Met Gly Ala Asp Gly Lys Met Gln Gly Leu 100 105110 Leu Gln Tyr Ser Glu Glu Asp Cys Ala Phe Glu Glu Glu Ile Arg Pro 115120 125 Asp Gly Tyr Asn Val Tyr Arg Ser Glu Lys His Arg Leu Pro Val Ser130 135 140 Leu Ser Ser Ala Lys Gln Arg Gln Leu Tyr Lys Asn Arg Gly PheLeu 145 150 155 160 Pro Leu Ser His Phe Leu Pro Met Leu Pro Met Val ProGlu Glu Pro 165 170 175 Glu Asp Leu Arg Gly His Leu Glu Ser Asp Met PheSer Ser Pro Leu 180 185 190 Glu Thr Asp Ser Met Asp Pro Phe Gly Leu ValThr Gly Leu Glu Ala 195 200 205 Val Arg Ser Pro Ser Phe Glu Lys 210 21511 10 PRT Unknown Residues which contain the anitgenic determinantrecognized by the myc monoclonal antibody. 11 Glu Gln Lys Leu Ile SerGlu Glu Asp Leu 1 5 10 12 5 PRT Unknown Preferred thrombin cleave site.12 Leu Val Pro Arg Gly 1 5 13 10 PRT Unknown Residues which bind toparamagnetic streptavidin beads (used for purification). 13 Ser Ala TrpArg His Pro Gln Phe Gly Gly 1 5 10 14 7 PRT Homo sapiens 14 Arg Pro TyrAsp Gly Tyr Asn 1 5 15 6 PRT Homo sapiens 15 Leu Pro Met Leu Pro Met 1 516 6 PRT Homo sapiens 16 Arg Pro Asp Gly Tyr Asn 1 5 17 6 PRT Homosapiens 17 His Phe Leu Pro Met Leu 1 5

We claim:
 1. An isolated nucleic acid molecule comprising apolynucleotide, wherein said polynucleotide encodes SEQ ID NO:4 and hasthe nucleotide sequence of SEQ ID NO:3.
 2. The isolated nucleic acidmolecule of claim 1, which is DNA.
 3. A method of making a recombinantvector comprising inserting a nucleic acid molecule of claim 1 into avector in operable linkage to a promoter.
 4. A recombinant produced bythe method of claim
 3. 5. A method of making a recombinant host cellcomprising introducing the recombinant vector of claim 4 into a hostcell.
 6. An isolated recombinant host cell produced by the method ofclaim
 5. 7. A recombinant method of producing a polypeptide, comprisingculturing the recombinant host cell of claim 6 under conditions suchthat said polypeptide is expressed and recovering said polypeptide.
 8. Akit for detecting the presence of mRNA encoding FGF-21 in a sample froma patient, said kit consisting of the polynucleotide of claim 1,packaged in a container.